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United States Department of Agriculture

Agricultural Research Service

Research Project: PROTOZOAN PARASITES AFFECTING FOOD ANIMALS, FOOD SAFETY, AND PUBLIC HEALTH
2008 Annual Report


1a.Objectives (from AD-416)
The objectives are to improve food safety and reduce contamination of drinking water by improving detection, determining sources, and reducing transmission of protozoan parasites infecting humans. Objective 1 a) Improve speed and accuracy of methods to detect Cryptosporidium, Giardia, and Microsporidia in selected environmental specimens and in specimens from food animals, other farm animals, wildlife, and transport hosts that might harbor multiple species or genotypes. b) Develop monoclonal antibodies specifically to identify zoonotic species of Cryptosporidium.

Objective 2 a) Determine the prevalence Blastocystis spp. in 1000 pre- and post-weaned dairy cattle from farms in eastern states utilizing DNA from our immediate past project; determine the prevalence of Microsporidia, Blastocystis, Giardia, and Cryptosporidium in 150 sheep and 500 pigs from birth to market age from multiple farms and states, and from 1000 feedlot beef cattle in Nebraska. b) Determine the presence of these same organisms in environmental specimens provided by NOAA collaborators from waters impacted by agricultural runoff. c) Assess the potential infectivity, duration of infection, and numbers of parasites excreted throughout a period of infection, by experimentally infecting parasite-free cattle, sheep, pigs, chickens, turkeys, and laboratory rodents with any unique genetic isolates found in the field studies described above.

Objective 3 a) Test for protective immunity of HBC fed to neonatal calves experimentally challenged with C. parvum oocysts by observing the severity and duration of infection. b) Conduct biochemical and molecular studies that might serve as a basis for future treatment strategies to interfere with transmission of parasites. c) Test anti-viral drugs associated with reduction of cryptosporidiosis in AIDS patients and in vitro will be tested for efficacy against zoonotic Giardia and Cryptosporidium, both of which have been shown to contain RNA viruses.


1b.Approach (from AD-416)
Studies will identify Giardia, Cryptosporidium, and Microsporidia of livestock and wildlife by developing multiplex PCR techniques and examining new gene sequences to provide improved characterization of these organisms. Viruses have been found within Giardia and Cryptosporidium, and studies will determine if differences in the quality or quantity of such viruses using newly developed reagents can facilitate detection and differentiate pathogenic and non-pathogenic strains. The prevalence of Giardia, Cryptosporidium, Microsporidia, and Blastocystis in sheep and pigs, and feedlot cattle will be determined. The prevalence of Blastocystis also will be determined in dairy cattle. Unique genotypes of these pathogens from field isolates will be tested in transmission studies to determine their potential to infect other animal hosts. The presence of zoonotic protozoan pathogens in environmental specimens in areas impacted by runoff from agricultural animals will be assessed. Studies will identify methods to provide protective immunity against Cryptosporidium. Cows will be immunized with recombinant proteins and immune stimulators to produce colostrum with high levels of anti-Cryptosporidium antibody for passive immunization of calves. Biochemical and molecular techniques will be used to study encystation/excystation in Giardia and Cryptosporidium to identify proteins that can be targeted to disrupt transmission. Anti-viral and anti-protozoal drugs will be tested against Cryptosporidium and Giardia using cell culture and animal infectivity models.


3.Progress Report
A multiplex PCR test was developed to detect the major species of Cryptosporidium that infect humans and cattle and differentiate these from minor or noninfectious species. A manuscript is in progress. Monoclonal antibodies produced against virus-like particles (CPV) found within the fecal stage of Cryptosporidium parvum and reverse transcriptase polymerase chain reaction for the CPV indicated that a greater concentration of CPV particles was associated with an isolate of C. parvum that produced more pathogenesis and greater numbers of excreted parasites in dairy calves. Results were published. Feces from a commercial flock of lambs and ewes, examined by molecular methods contained a genotype of Cryptosporidium infectious for humans as well as species and genotypes that infect only sheep. Findings were published on the intragenic variation of this genotype. DNA extracted from feces of dairy cattle from birth to 2 years of age was examined for the presence of Blastocystis, a pathogenic microorganism of humans and animals. Although only about one third of the DNA specimens have been examined Blastocystis has been detected for the first time in North American dairy cattle. In cooperation with APHIS feces from feedlot cattle in central and western states are being examined for the presence of Cryptosporidium. Nucleoside drugs are being tested in cell culture to determine if they have any effect against the CPV particles found within Cryptosporidium. Using previously developed cDNA libraries enriched for sequences specific for Giardia cysts and trophozoites and another cDNA library enriched for specific sequences for Cryptosporidium oocysts, sequences of activated genes (EST’s) from Giardia DNA libraries are being analyzed and deposited into GenBank. Ongoing subtractive hybridization studies will determine how these parasites form protective cysts that enable them to survive in the environment and how they are released from cysts after entering the body. SYs and technicians meet weekly on Monday morning to discuss findings from the previous week and to make plans for the coming week. Under National Program 108 (Food Safety) the following Component 1 topics apply to the current research plan: 1.1 Pathogens, Toxins and Chemical Contaminants Preharvest, 1.1.1 Methodology, 1.1.2 Epidemiology, 1.1.3 Ecology, Host Pathogen and Chemical Contaminants Relationships, and 1.1.4 Intervention Strategies.


4.Accomplishments
1. Blastocystis sp., an enigmatic widespread microbe has been recorded from humans, pigs, birds, rodents, amphibians, reptiles, and fish. Little is known about how it is transmitted to humans. However, very recently it has been identified by molecular methods to consist of numerous unique genetic types found in different host species. We have detected Blastocystis in the feces of dairy cattle for the first time in North America. The only report of Blastocystis in dairy cattle has been from a few Japanese cattle. Addresses Program 108 objectives specifically related to action plan component 1.1.1: sampling, isolation, identification and quantification of pathogens in animal fluids and tissues, manure; and the environment, including feed, water, and wild animals.


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of the New MTAs (providing only)4
Number of Non-Peer Reviewed Presentations and Proceedings13

Review Publications
Fayer, R. 2008. The General Biology of Cryptosporidium. In: Fayer, R., Xiao, L., editors. Cryptosporidium and Cryptosporidiosis. 2nd edition. Boca Raton, FL: CRC Press. p.1-42.

Fayer, R., Xiao, L. 2008. Cryptosporidium and Cryptosporidiosis. Cryptosporidium and Cryptosporidiosis. Boca Raton, FL. CRC Press. 560 p.

Santin, M., Trout, J.M., Fayer, R. 2008. A longitudinal study of Cryptosporidiosis in dairy cattle from birth to two years of age. Veterinary Parasitology. 155(2008):15-23.

Trout, J.M., Santin, M., Fayer, R. 2008. Detection of Assemblage A, Giardia duodenalis and Eimeria spp. in Alpacas on Two Maryland Farms. Veterinary Parasitology. 153:203-208.

Fayer, R., Fair, P.A., Bossart, G.D., Santin, M. 2008. Examination of Naturally-Exposed Bottlenose Dolphins (Tursiops truncatus) for Microsporidia, Cryptosporidium, and Giarda. Journal of Parasitology. 94:143-147.

Fayer, R., Santin, M., Trout, J.M. 2007. Enterocytozoon bieneusi in mature dairy cattle on farms in the eastern United States. Parasitology Research. 102(1):15-20. Epub 2007 Sep 27.

Feng, Y., Ortega, Y., He, G., Das, P., Xu, M., Zhang, X., Fayer, R., Gatei, W., Cama, V., Xiao, L. 2007. Wide distribution of Cryptosporidium bovis and the deer-like genotype in bovines. Veterinary Parasitology. 144:1-9.

Jenkins, M.C., Higgins, J.A., Brahante, J., Kniel, K., Obrien, C.N., Trout, J.M., Lancto, C., Abrahamsen, M., Fayer, R. 2008. Fecundity of Cryptosporidium parvum is Correlated with Intracellular Levels of the Viral Symbiont CPV. International Journal for Parasitology. 38:1051-1055.

Santin, M., Trout, J.M. 2008. Cryptosporidiosis of Companion Animals. In: Fayer, R. Xiao, L., editors. Cryptosporidium and Cryptosporidiosis. 2nd edition. Boca Raton, FL: CRC Press. p. 437-449.

Santin, M., Trout, J.M. 2008. Cryptosporidiosis of Livestock. In: Fayer, R., Xiao, L., editors. Cryptospiridium and Cryptosporidiosis. 2nd edition. Boca Raton, FL. CRC Press. p. 451-483.

Xiao, L., Fayer, R. 2008. Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission. International Journal for Parasitology. 38(2008):1239-1255.

Santin, M., Cortes Vecino, J.A., Fayer, R. 2008. Enterocytozoon bieneusi genotypes in dogs in Bogota, Colombia. American Society of Tropical Medicine and Hygiene. 79(2):215-217.

Last Modified: 9/21/2014
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